ABSTRACT Infections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) during pregnancy are associated with premature rupture of membranes, preterm labor and delivery, low birth weight, congenital infections, perinatal death and mother-to-child transmission of HIV infection. Sexually transmitted infections (STIs) like these are common in pregnant women globally, but often go undiagnosed; recent work by our group found a 41% STI prevalence amongst HIV-infected pregnant women, of which 64% of infections were asymptomatic. Recent research suggests the vaginal microbiome may play a critical role in STI acquisition, persistence and treatment outcomes. Our pilot work has shown that diagnostic testing for CT, NG, and TV in antenatal care services for HIV-infected pregnant women in South Africa is highly acceptable and feasible; however, our work has made clear that evaluating the impact and cost effectiveness of different diagnostic screening strategies that optimally decrease the burden of STIs during pregnancy and at time-of-delivery is urgently needed. Furthermore, our findings highlight that biological factors that increase the risk for STI persistence and/or treatment failures must be further investigated. In response to the need to 1) identify optimal, cost-effective screening strategies that decrease the burden of STIs during pregnancy and reduce adverse birth outcomes, 2) provide evidence to update WHO's syndromic management guidelines, and 3) elucidate the role of the vaginal microbiome in STI treatment outcomes, we propose a novel, highly innovative study with the following three Aims: Aim 1: Evaluate 3 different screening strategies to decrease the burden of CT/NG/TV among pregnant women, and reduce adverse birth outcomes. Aim 2: Evaluate cost per pregnant woman screened and treated, cost of adverse birth outcomes, and cost-effectiveness per STI and disability-adjusted life-year (DALY) averted. Aim 3: Investigate the relationship between the vaginal microbiome and persistent Chlamydial infections in pregnant women. Our proposed 5-year study will enroll 1250 HIV-infected and 1250 uninfected pregnant women from three large ANC clinics in Tshwane District, South Africa, as well as their ~2500 neonates and up to 834 male partners. Our research team, led by established researchers, has significant expertise and experience in all aspects of the proposed study. Our multi-institutional collaborations will allow us to leverage unique implementation platforms and resources, and allow for rapid dissemination of findings to South African and global stakeholders.